Method and device

ABSTRACT

A method of modification of a protein or polypeptide in the presence of a modifying composition capable of providing at least one modification wherein a liquid phase comprising the protein or polypeptide is brought into contact with a solid phase capable of immobilizing the protein or polypeptide and the solid phase carrying the immobilized protein is brought at least once into contact with a liquid phase comprising the composition capable of modifying the protein or polypeptide and modification reaction(s) are allowed to occur. The liquid phase comprising the protein or polypeptide may be a liquid extract of eukaryote or prokaryote cells. The modification may be a acylation, phosphorylation, dephosphorylation, SUMOylation, ubiquitinylation, carboxymethylation, formylation, acetylation, deacetylation, gamma carboxyglutamic acid, norleucine, amidation, deamidation, carboxylation, carboxyamylation, sulfation, methylation, demethylation, hydroxylation, ADP-ribosylation, maturation, adenylation, O-linked glycosylation, N-linked glycosylation, methonine oxidation, and addition of lipid (prenylation).

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a method of chemical or enzymaticmodification of proteins and polypeptides and to a kit for use in such amethod. In particular it relates to a method of purification andpost-translational modification (PTM) of proteins and polypeptides andto a kit for use in this method.

BACKGROUND OF THE INVENTION

Within the field of life science, there is an increasing recognition ofthe importance of post-translational modifications of proteins ineukaryotic cells. Many modified proteins are critical in cell signalingprocesses or crucial to biological processes and are often modified inhighly specific sub-cellular localizations or as a function of temporalgradient or timing event. However, modified proteins are very hard toidentify and isolate from endogenous material. This difficult isolationof a homogeneously modified target protein increases the difficulty topurify this form of protein and dramatically decreases the yield.

In view of this, a major problem with conventional cellularcharacterization studies is the definition or isolation ofunderrepresented or low-abundance proteins and their differentialexpression patterns, often comprising post-translational proteinmodifications. It is these difficult to characterize proteins that areoften the most interesting to understand and confer a functionalcharacteristic of a specific role in the cell. The production of thesemodified proteins can often be seen as being critical to understandingtheir function, or dysfunction, in a biological system.

Also, in some functional studies and/or diagnostic assays, it isbecoming apparent through experimental observations that modifiedproteins can be considered essential. In terms of recombinant material,often these proteins are produced without modification and functionpoorly compared to their modified form, whereas, as stated herein above,the isolation of endogenous modified forms of the protein can beextremely difficult and yield low levels in terms of quantity and poorlevels in terms of purity and homogeneity.

For functional studies and/or diagnostic assays, post-translationallymodified proteins provide a tuning that cells need to function.Producing modified proteins are critical to understanding complexprotein mediated pathways. There are often not enzymatic intermediatesto follow signaling pathways and events, but protein-proteininteractions mediating or modulating a certain effect in the cell. It isin these cases that post-translational modifications are becomingevident to be playing increasingly complex roles in controlling andsignaling biological systems.

A severe problem in the field of structural studies of modified proteinsis due to the amount of material needed and the level of purity neededto conduct an experiment. Large quantities of highly purified,homogenous material are necessary to conduct a structural study. Theproduction of suitable material is often the rate-limiting step. Theproduction of eukaryotic modified material is expensive, inefficient andoften results in a highly heterogeneous product.

Within the field of drug screening and production, the use ofrecombinant material is an attractive alternative considering the costand requirement of large quantities of material needed to study. Adrawback is the form of material produced, which generally fails toprovide the eukaryotic like modifications that can best mimic the systemthat the drugs are being designed and intended in use for, oftenmammalian systems. This is a problem especially in view of the fact thattypical drug-screening campaigns are expensive and that the success ofthe assay often depends on the quality of the target protein being used.In addition, this applies to diagnostic assays and screens that may beused as analytical tools or in high-throughput screening applications.

Also in the actual production of recombinant peptides for the use asdrugs, post-translational modifications (such as amidation, necessaryfor amino and/or carboxy-terminal protection) are often required in theformulation of that product.

Marcucci et al., in U.S. Pat. No. 6,172,202 disclose a process for thepreparation of a conjugate between a poly (ethylene glycol) (PEG) and aprotein or glycoprotein comprising specifically binding the domain ofthe protein or glycoprotein to a specific binder to shield it from thePEG in the conjugating step, and wherein the specific binder is releasedsubsequent to the conjugation. The advantage achieved by the process ofMarcucci et al is said to be a more homogeneous product and preservingof biological activity. No mention is made of immobilization of aprotein or of enzymatic modification of a protein.

Dalborg et al., in U.S. Pat. No. 6,048,720 disclose a process forimproving the in vivo function of factor VII by shielding exposedtargets of the same, comprising immobilizing factor VII by interactionwith a group-specific adsorbent and subsequently conjugating anactivated biocompatible polymer, preferably a PEG homopolymer, to theexposed targets of the immobilized factor VII. The purpose of theimmobilization is to exclude the interaction sites on the polypeptidefrom conjugation to the biocompatible polymer.

It seems that Dalborg et al. and Marcucci et al. generally address thesame problem of conjugating a polymer, basically a PEG polymer, to apolypeptide whilst shielding the active site of the latter from theconjugation reaction.

Colpan et al., in DE 3717210A1, disclose a method of modifying abiopolymer by immobilizing it on an adsorbent, reacting the immobilizedbiopolymer, optionally use it further and desorbing the reactionproduct. It appears that the biopolymer to be modified by method ofColpan et al. is a nucleic acid. The immobilization of nucleic acids andmodification of nucleic acids is quite different from protein/peptideisolations and modifications. Nucleic acids are very robust molecules(biopolymers) that can be subjected to a wide range of experimentalconditions, whereas proteins/peptides are vulnerable and sensitive undera similar range of conditions. The nucleic acid method described byColpan et al. involves completely denatured nucleic acid materialincompatible with chemically and biologically relevant conditions.

T. P. Bradshaw and R. B. Dunlap in Biochemistry, 32 (1993) 12774-12781investigate a heterodimer of thymidylate synthase using chemicalmodification of the enzyme immobilized on a Sepharose resin. Theirmethod however is very inefficient, requiring several purification stepsand providing only a low yield. The aim of the method is apparently onlyfor analytical biochemical analysis and no suggestion is made of anyadvantageous modified protein production.

WO 00/50902 discloses a method for analysing a sample containing“biologically significant molecules”, such as enzymes and enzymemodulators, which generally consists of providing a pair of proteins orpolypeptides capable of associating with one another in a way dependenton the state of modification of at least one of the two entities in thepair, one of them being immobilized on a solid phase. The modification,under the action of the “biologically significant molecules” in thesample, of any the two proteins/polypeptides is susceptible ofinfluencing the association of said proteins/polypeptides. The state ofassociation therefore is used a means for assessing the modificationwhich has taken place. In said method, use is made of a pure andwell-known protein composition as a means for studying e.g. enzymes andenzyme modulators and samples susceptible to contain them.

The methods of the prior art as summarized herein above, when directedto the production of proteins, all teach performing an important numberof purification steps on the protein and suffer from low yield.

SUMMARY OF THE INVENTION

In a first aspect of the present invention there is provided a method ofchemical or enzymatic modification of a protein or polypeptide asspecified in claim 1.

According to a still further aspect of the invention, kits are providedfor use in any of the inventive methods and/or applications.

Further aspects of the invention are as defined in the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

To facilitate understanding of the invention, exemplary embodiments willbe described further below, with reference to the drawings, wherein:

FIG. 1 represents immobilization of a protein or polypeptide on a solidphase.

FIG. 2 represents modification in-line on-column of protein orpolypeptide in the presence of modifying composition.

FIG. 3 represents modification in-line on-column-reiterated steps.

FIG. 4 represents cleavage of suitably modified protein or polypeptidefrom fusion linker.

FIG. 5 represents elution of modified protein or polypeptide.

FIG. 6 represents purification of rat HNF4α LBD::GST fusion protein on aGSTPrep 16/10 column.

FIG. 7 represents purification of rat HNF4α LBD on a Resource Q 6 mlcolumn.

FIG. 8 represents purification of rat HNF4α LBD on a HiLoad 16/60Superdex 75 prep grade column.

FIG. 9 represents SDS-PAGE analysis of the protein samples from ratHNF4α LBD purification.

FIG. 10A represents MALDI-TOF MS identification of the purified ratHNF4α LBD-m/z spectrum.

FIG. 10B represents the full length amino acid sequence of the rat HNF4αprotein.

FIG. 11 represents purification of mouse TDG on HiPrep Heparin 16/10column.

FIG. 12 represents purification of mouse GST::TDG fusion protein on aGSTPrep 16/10 column.

FIG. 13 represents purification of mouse TDG on HiLoad 16/60 Superdex200 prep grade column.

FIG. 14 represents SDS-PAGE analysis of the protein samples from mouseTDG purification.

DETAILED DESCRIPTION OF THE INVENTION

In one important aspect the invention aims at providing a methodallowing for the purification, post-translational modification andisolation of a modified protein or polypeptide in a systematic,reproducible and effective manner.

Furthermore, in another aspect, the present invention aims at providinga method allowing for the scale-up of post-translationally modifiedprotein or polypeptide production to obtain large amounts of modifiedmaterial that has the distinct advantage of higher yields and higherlevels of purity which, by following this method, can be used forproduction, analytical and large-scale industrial applications.

Moreover, the present invention aims at providing a method allowing foran extensively automated production of post-translationally modifiedproteins or polypeptides.

Generally, a method of the invention will comprise the steps of

-   i) bringing a liquid phase comprising the protein or polypeptide    into contact with a solid phase upon which the protein or    polypeptide is susceptible of being immobilized and allowing    immobilization of the protein or polypeptide on the solid phase to    occur; and-   ii) bringing the solid phase carrying the immobilized protein or    polypeptide into contact with solution comprising at least one    modifying enzyme, susceptible of catalyzing a modification reaction    of the protein or polypeptide, and any other components necessary    for the modification reaction(s) and allowing the modification    reaction(s) to occur.

The source of the protein or polypeptide is not critical to theinvention. For example, using conventional genetic engineeringtechniques, the protein or polypeptide may be produced by a hostorganism, e.g. a yeast cell or a bacterium such as Escheerichia coli orBacillus subtilis, which has been transformed or transfected with anexpression vector, obtained by insertion of a coding gene or partthereof into a vector in a conventional manner. The transformed ortransfected host is cultured and proliferated under suitable conditions,as known to the person skilled in the art. However, it should beunderstood that the invention is by no means limited to recombinantlyproduced proteins or polypeptides, but could be applied in principle toany protein obtained from any source.

The way of obtaining a liquid phase comprising the protein orpolypeptide also is not critical to the invention. For example, theprotein or polypeptide may be present in a liquid phase containing acellular extract from a eukaryote or prokaryote cell, e.g. a bacterialhost over-expressing a recombinant protein or polypeptide. The cellularextract may be obtained by methods well known to the person skilled inthe art, e.g. by cell lysis, centrifugation, ultracentrifugation,collecting of supernatant fractions etc. It is a very advantageousfeature of the invention that the method may be applied directly to acellular extract having been subjected to no purification step. Indeed,in most of the methods of the prior art, purification of cellularextract are required leading to substantial losses and decrease ofoverall yield. In contrast, in the method of the invention the proteinin the cellular extract may be selectively immobilized on the solidphase, the other, contaminating components washed away, and themodification may be performed.

The liquid phase comprising the protein or polypeptide may be a suitableaqueous buffer solution, such as PBS (Phosphate Buffered Saline), HEPES(4-(2-Hydroxyethyl)-1-piperazineethanesulfonic Acid), Tris(Tris[hydroxym ethyl]aminomethane) and MES(2-[N-Morpholino]ethanesulphonic acid).

This liquid phase containing the protein or polypeptide to bepost-translationally modified is brought into contact with a solidphase, upon which the protein or polypeptide selectively is retained.The solid phase may be any suitable matrix, e.g. a resin, such as anaffinity resin or ion exchange resin, upon which the protein orpolypeptide is immobilized by a characteristic of its nature or by beingfused to a fusion linker/moiety.

In a preferred embodiment, at least one exchange of liquid phase incontact with the solid phase carrying the immobilized protein orpolypeptide is then performed: this step may comprise bringing the solidphase carrying the immobilized protein or polypeptide in contact with aliquid phase, of the same basic composition as the original liquid phasecomprising the target protein or polypeptide, or of a differentcomposition. Exchanging the liquid phase advantageously improves theenrichment level and homogeneity of the immobilized protein orpolypeptide, removing further contaminants. In addition, with theforesight of knowing what the downstream applications are, the liquidphase can be exchanged to be compatible with downstream processes (e.g.remove phosphate component in a binding buffer to exchange withTris-based buffer, remove additives, detergent exchange, introduceadditives essential for downstream process such as reducing agents,salts, etc.). This step has the advantage of also completing anadditional washing step enriching the immobilized target protein orpolypeptide further. This step can be reiterated depending on thedownstream process (multiple phosphorylation steps with differentkinases under different conditions, multiple modifications, on-columncleavage reactions, etc.).

In the subsequent step ii), the solid phase carrying the immobilizedprotein or polypeptide is brought into contact with a liquid phasecomprising at least one modifying enzyme and any other componentsnecessary for the modification reaction(s) to occur, and then allowingthe modification reaction(s) to occur.

Exemplary for components which are necessary for the modificationreaction(s) to occur are reducing agents, co-factors, substrates etc.,such as ATP (adenosine 5′-triphosphate; nucleotide donor), EDTA(Ethylene Diamine Tetraacetic Acid; metal ion chelator), phosphatidylserine (activating co-factor) and detergents (Triton-X-100, NP40,dodecylmaltoside; which increase protein or polypeptide stability andsolubility).

The liquid phase per se may be of the same kind as the precedent liquidphase(s) or may differ therefrom. It preferably comprises buffersolution plus additional components that increase the efficiency of themodification reaction.

The modification reaction may be performed in batch or in-lineon-column, and can be stringently controlled. To ensure reproducibility,the incubation time and temperature may be monitored, and all thereaction components to be supplemented systematically introduced. Theprocess may be automated.

In one advantageous embodiment, the reaction, also termed incubation, isperformed in-line on-column. This allows for time-dependent incubationfor post-translational modifications to the immobilized target proteinor polypeptide. In addition, for fusion proteins or polypeptides thatrequire proteolytic cleavage to remove the fusion tag (either N- orC-terminal fusions with a (proteolytic) cleavage site), this step can beused to process the immobilized fusion protein or polypeptide byremoving the affinity tag.

In one embodiment, the liquid phase comprising enzyme is circulated,i.e. the solid phase solid phase carrying the immobilized protein orpolypeptide is brought into contact therewith more than once, e.g. from2 times, or by use of a recirculation-loop providing constant modifyinginteractions for a given time frame and flow rate variables. This allowsfor a shorter total incubation time since the diffusion of enzyme andother components within the liquid phase to the immobilized protein orpolypeptide becomes less important as a rate limiting parameter. Thenumber of circulations may be selected so as to obtain a suitable yieldof the modification process.

Next, the modifying enzyme(s) and auxiliary components may be washedaway, by bringing the solid phase carrying the modified protein orpolypeptide in contact with a suitable liquid phase. This step can workas a buffer exchange, but can also be used as a further purificationenrichment step to increase the purity of the immobilized target proteinor polypeptide. All unwanted reaction components may be washed awaywhereas the immobilized modified protein or polypeptide remains bound tothe resin.

In one embodiment, a liquid phase comprising modifying enzyme(s) andauxiliary components of a different kind is subsequently brought intocontact with the solid phase carrying the still immobilized protein orpolypeptide, which will comprise modified as well as non-modifiedprotein or polypeptide, and the protein or polypeptide is furthermodified.

This sequence of successive modifications can be performed in principleas many times as desired and may be optimized to obtain a given desiredyield of a protein or polypeptide suitably modified.

Subsequently, the modified protein or polypeptide may be collected fromthe solid phase by an elution step using a suitably selected liquidphase, such as a suitable buffer solution. Advantageously, the liquidelution phase may be selected in view of the downstream events, such asthe projected use of the modified protein or polypeptide. Subsequent toelution, modified protein(s) or polypeptide(s) may be separated fromnon-modified proteins or polypeptide(s) by any conventional protein orpolypeptide separation method, known to the person skilled in the art,such as analytical gel filtration, or other chromatographic methodsseparating modified vs. unmodified proteins or polypeptides. Theseauxiliary steps can be used to approach the highest level of modifiedprotein enrichment (highly pure modified target protein). In principle,the homogeneity of the modification reaction is completed to its highestlevel prior to elution from the immobilized status.

Some exemplary embodiments of the invention will now be brieflyoutlined, referring to the figures. In FIG. 1 a protein or polypeptide1, fused to a fusion linker 2, is immobilized on a resin by affinity offusion linker 2 for ligand group 3 on the resin. In exemplaryalternative embodiments, the protein or polypeptide 2 may be immobilizedby a characteristic of its nature (ex. immuno-affinity, functionaldomain (nucleotide or heparin binding), or chemical nature (ionexchange)). The immobilized protein or polypeptide 2 can then beenriched further removing contaminants by use of binding buffer.

Buffer exchange preferably is then performed to improve the enrichmentlevel and homogeneity of the immobilized protein or polypeptide 2 byremoving contaminants. Optionally, the buffer is exchanged to becompatible with downstream processes This step may be reiterated, suchas for multiple modification steps with different modifying componentsunder different conditions, on-column cleavage reactions, etc. Reactionbuffer and modifying component(s) 4 are added (FIG. 2), to provide formodification X of the protein. The incubation is performed in batch orin-line on-column, whereby the X modification reaction may bestringently controlled by appropriate monitoring of the incubation time,temperature, and all the reaction components to be supplemented.

The immobilized X-modified protein or polypeptide 2 may be submitted toZ-modification using modifying component 4′ (FIG. 3) optionallysubsequent to buffer exchange and/or purification enrichment increasingthe purity of the immobilized X-modified protein 1 or polypeptide.

The preceding steps can be reiterated in a cyclic manner for a number ofmodifications to a target protein or polypeptide, whereby all unwantedreaction components are washed away and the immobilized modified targetremains bound to the resin.

To collect the suitably X,Y-modified protein or polypeptide 1 withoutthe fusion linker 2, a cleavage component 5 for breaking the bondbetween these is added, such as a protease (FIG. 4).

Finally, the X,Y-modified and cleaved protein or polypeptide iscollected by elution from the resin (FIG. 5).

The yield of modified protein or polypeptide can be assayed by severalanalytical methods, known to the person skilled in the art, such aselectrophoresis (native and denaturing), immunoblotting, massspectrometry, functional/activity assays, NMR, and crystallography. Thefinal product is highly enriched, highly pure, post-translationallymodified, in high yield and concentration.

When the protein or polypeptide is fused to a fusion linker, i.e. aprotein or peptide fragment having affinity for the solid phase, theelution step comprises bringing the solid phase carrying the modifiedfused protein or polypeptide into contact with a liquid phase comprisingan enzyme susceptible of cleaving the bond between the modified proteinor polypeptide and the protein or peptide fragment having affinity forthe solid phase as well as any other components necessary for thecleaving reaction to occur.

In principle, any type of protein or polypeptide modifications,enzyme-catalyzed or not, may be performed by the method of theinvention, such as e.g. a acylation, phosphorylation, dephosphorylation,SUMOylation, ubiquitinylation, carboxymethylation, formylation,acetylation, deacetylation, gamma carboxyglutamic acid, norleucine,amidation, deamidation, carboxylation, carboxyamylation, sulfation,methylation, demethylation, hydroxylation, ADP-ribosylation, maturation,adenylation, O-linked glycosylation, N-linked glycosylation, methonineoxidation, and addition of lipid (prenylation).

To illustrate the importance of post-translational modificationreactions in eukaryote organisms, some of them will be discussed hereinbelow. Phosphorylation Among all post-translational modifications in theeukaryote cell, phosphorylation is a frequently used and powerfulmechanism for the rapid modulation of transcription factors activity inresponse to environmental conditions and hormonal signals. Most, if notall, nuclear receptors studied to date are phosphoproteins whosefunctions are regulated by phosphorylation. In view of this, a methodallowing to produce precisely and reproducibly post-translationallyphosphorylated molecules will be of great advantage e.g. for thescreening of possible drugs, for the study of regulatory phenomena ofthe eukaryotic cell etc.

SUMOylation (Small Ubiquitin-Related Modifier)

A number of eukaryotic proteins are post-translationally modified by theubiquitin-like modifier SUMO-1 protein (small ubiquitin-relatedmodifier) and its close homologues SUMO-⅔. The pathway of SUMOylation ismechanistically analogous to ubiquitinylation, however requiring adistinct set of enzymes. Presently, the dimeric SUMO-activating enzyme(UBA2/AOS 1) and the SUMO-specific E2 enzyme UBC9 have beencharacterized.

The SUMO-1 protein functions in protein-protein interactions, signaltransduction, can act as an antagonist to ubiquitinylation and proteindegradation, increasing proteins stability and half-life.

Ubiquitinylation

In the process of ubiquitinylation a protein is modified by covalentbonding to ubiquitin through the ligation of the C-terminus of ubiquitinto the á-amino groups of protein lysine residues. Ubiquitin is a small,76-residue protein common to all eukaryote organisms. The major, but notsole, function of ubiquitinylation is to target proteins fordegradation, usually by formation of a multiubiquitin chain on thetarget protein. Recently, dysfunctional ubiquitin pathway ofoncoproteins has been recognized to be implicated in development oftumors.

Ubiquitinylation of specific lysine residues involves a multi-enzymesystem, but the key component in regulation and biological specificityis the ubiquitin-protein ligase (also known as E3).

Carboxymethylation/N-Ethylmaleimide (NEM)

This is a modification providing protection of solvent exposed cysteineresidues to prevent aggregation and oxidation of proteins. It isadvantageously used for the production of hydrophobic proteins that aresubject to oxidation-dependent aggregation and therefore, precipitation.It allows for the production and isolation, in a concentrated form, ofhydrophobic proteins that are known to aggregate and precipitate.

Acetylation

Acetylation is used to modify positively charged Lys/Arg amino acidresidues. It has an important function in chromosomal DNA remodeling andtranscriptional regulation. It is a reversible modification.

Amidation

This provides a means of amino and/or carboxy-terminal protection ofpolypeptides. This is valuable in drug production/bioprocessingapplications where the amino and/or carboxy-terminal must be protectedfor transport as a drug increasing stability. Many bioactive peptidesmust be amidated at their carboxy terminus to exhibit full activity. Thepeptides are synthesized from glycine-extended intermediates that aretransformed into active amidated hormones by oxidative cleavage of theglycine N—C alpha bond. In higher organisms, this reaction is catalyzedby a single bifunctional enzyme, peptidylglycine alpha-amidatingmonooxygenase (PAM). The PAM gene encodes for two enzymes that catalyzethe amidation reaction. Peptidylglycine alpha-hydroxylatingmonooxygenase catalyzes the stereospecific hydroxylation of the glycinealpha-carbon of all the peptidylglycine substrates.Peptidyl-alpha-hydroxyglycine alpha-amidating lyase generatesalpha-amidated peptide product and glyoxylate.

Methylation

Methylation is used to modify positively charged Lys/Arg amino acidresidues. It has an important function in chromosomal DNA remodeling andtranscriptional regulation and is a reversible modification.

Glycosylation

Glycosylation is used to modify proteins by bonding them tocarbohydrates (oligosaccharides). There are two main types; O-linlced(to the OH side chain of Ser and Thr) and N-linked (to the NH2 sidechain of Asn in the sequence Asn-X-Ser/Thr, where X can be any aminoacid besides Pro and Asp). Some proteins are attached to the plasmamembrane by a third type of carbohydrate structure called a glycosylphosphatidylinositol (GPI) anchor. Modification in vivo can only beperformed in the lumen of the rough endoplasmic reticulum, and cansubsequently be modified in the lumen of the golgi apparatus where otheramino acids of the protein may become glycosylated. Glycosylation is areversible modification.

The above enumerated and briefly outlined post-translationalmodifications are exemplary only, and the invention should not beconstrued as limited to these. More details about post-translationalmodifications of proteins may be found in T. E. Creighton, Proteins:Structures and Molecular Properties. W. H. Freeman and Company, NewYork, second edition. (1993) ISBN 0-7167-7030-X.

The method of the invention has several advantageous features: Firstly,it may be performed in one single chromatographic step. The method canbe performed in batch mode, in a self-packed column or in a pre-packedcolumn format.

The batch mode format can be applied in multi-well titre plates for highthroughput applications. It allows the users to use the resin of theirchoice, and it does not necessarily need the high-pressurecharacteristics of controlled flow systems (ex. chromatographicplatforms). Also, this can be applied directly on the lab bench withoutthe requisite of having a chromatographic platform. It facilitates thethroughput (doing multiple parallel experiments), and can allow forsimple experimental design and side-by-side comparisons for differentconditions. A method for screening for the immobilization, modificationand purification process of a, or series of, target(s) (and can also beused in immobilized/modified form for drug discovery approaches directlyscreening compounds on the immobilized modified targets on the resin).The batch format may be automated by integrating liquid handling robots,vacuum manifolds, and multi-well plate format injectors, chromatographicplatforms and sample collectors.

Self-packed columns allow the users to make columns of custom designedsize and capacity and to their own requirements. The use of self-packedcolumns is often for speciality resins that may not be available inpre-packed or batch format.

Pre-packed columns allow the users to purchase columns commerciallypacked or validated that have common parameters and functionality. Thequality control is very high and the reproducibility from column tocolumn is an asset. The ease of use and comprehensive data filesaccompanying the column make for simple and easy integration and usedirectly off the shelf. In addition, the systems are easy to adapt to awide range of chromatographic platforms. Furthermore, the “bioprocess”validation that is possible with many columns ensures that the columnmay be used in drug production processes.

A powerful advantage to this method is the coupling of affinitypurification to modification to on-column cleavage in an automated,systematic and reproducing manner on existing chromatographic platforms(ÄKTA™ or Ettan™ systems ex Amersham Biosciences of Uppsala, Sweden)producing a protein or polypeptide product that optionally has noaffinity fusion tag following modification and cleavage. This has manyadvantages such as more ‘native’ like form (in the case of no fusion tagpresent) and isolation of modified target protein or polypeptide only,no contamination of fusion protein.

Secondly, when applied to recombinant proteins or polypeptides, producedin prokaryotic cells, it allows for the production of a protein orpolypeptide close to the eukaryotic form: the modifications done toimmobilized target protein or polypeptide produce eukaryotic-likemodifications currently not possible to systematically control in vivoor under poor control and limited enrichment using in vitro modificationsystems.

Thirdly, a flexible method is provided which functions for a wide rangeof proteins or polypeptides, such as e.g. nuclear, cytosolic andmembrane proteins, proteins differing in chemical and/or functionalnature, enzymes, nucleic acid (RNA and DNA) binding proteins andcomplexes, immunoproteins, structural proteins and signaling proteins aswell as corresponding polypeptides.

In view of the temperature dependence of the kinetics of the enzymaticreaction, prior art methods for enzymatic reactions are generallyperformed at temperatures around 37° C. A decrease of the temperaturewould result in an increase of the reaction time, which could becomeunacceptably long. On the other hand, at the temperature required by theprior art methods, the stability of the protein and the polypeptide issusceptible of being deteriorated. This may be due both to increasedunfolding and misfolding of the protein and polypeptide with anincreasing temperature and to an increased risk of degradation reactionto occur, e.g. as catalyzed by any trace contamination of proteases. Anadvantageous feature according to the present invention is that theentire process can be performed at an appropriately selectedtemperature, which may be held constant or varied as required. Forexample, the condensed phases may be maintained at ambient temperature(about 25° C.). More preferably, the method of the invention isperformed at a temperature from 15° C. to as low 2° C., a mostpreferable temperature being 4° C. At this low temperature a slowerreaction due to temperature dependence may be compensated for byrepeatedly bringing the enzyme preparation into contact with the solidphase carrying the immobilized protein or polypeptide, such as byrecirculating continuously a liquid flow containing the enzymaticpreparation. By the inventive method the repeated circulation of themodification component, be it chemical or enzymatic, into contact withthe protein or polypeptide immobilized on the solid phase will allow fora relatively short total time of modification even at a reactiontemperature where, by the prior art methods, an unacceptably longduration of reaction period would be required.

As a further advantage of the low temperature of the modificationreaction, a further enhanced specificity of reaction is obtained, e.g.due to the fact that the enzyme at a lower temperature is lesssusceptible of spurious reactions.

The method of the invention advantageously may be performed in anautomated high-throughput experimental design, using chromatographicplatforms and columns such as the ÄKTA™ and Ettan™ Platforms, fromAmersham Biosciences of Uppsala, Sweden, but is not limited to the useon these specific platforms, or to the scale of these platforms.

The method, e.g. as performed on an ÄKTA platform for separation andmodification, can be scaled linearly (approximately) from the microlitre(protein or polypeptide production measured in growth media) to 100's oflitre scale. The method can also be applied using the Ettan platform forcoupling of the modification to identification and characterization ofmodification by mass spectrometry. This may be a valuable tool e.g. whenthe downstream event is functional genomics, drug target screeningapplications or applications where highly specific data is requiredconcerning the characterization of the modified target protein orpolypeptide.

In relation to drug discovery and drug screening, the method of theinvention provides a very important improvement. By use of a method ofthe invention the eukaryote protein or polypeptide of interest, e.g.associated to a disease, may be recombinantly produced in a suitableoverexpressing host, such as a bacterial strain, whereafter it isprecisely and selectively modified to similarity with the nativeeukaryote protein or polypeptide. This protein or polypeptide may beused, either in the immobilized state or after collecting it from thesolid phase, for efficient screening of substances for use as potentialdrugs.

According to one aspect of the invention, in relation to potential drugdiscovery, proteins or polypeptides, post-translationally modified by amethod according to the invention, may be used to raise antibodies forthe use in treating diseases or in the case of profiling or detectingdisease, or the onset of certain disease states.

Accordingly, a PTM biomolecule may be produced by modification of thecorresponding non-PTM biomolecule, and the PTM biomolecule may the beused in generating or raising antibodies against that specificmodification, relative to its unmodified parental biomolecule.

The antibodies may be used to treat diseases with specificantibody-antigen recognition and biological implications, or asdetection or profiling screens to study the onset or possible onset ofdisease in a diagnostic and/or screening type approach.

In an advantageous embodiment, the solid phase is an affinity resin towhich the target protein or polypeptide is immobilized by means of afusion linker. This may be achieved by manipulating, at the gene level,a target protein or polypeptide by fusing it to another protein orpeptide fragment, to obtain a so-called fusion protein or polypeptide.At the protein expression level, fusion proteins or polypeptides canhave the advantage of providing a more favorable gene constructorganization permitting higher levels of soluble protein or polypeptideto be expressed, and possibly reducing the propensity to drive theprotein or polypeptide folding process towards creating inclusionbodies. However, an inherent problem with the fusion protein/peptidesystem is that the ‘tag’ is often difficult to remove. Specificproteases required to perform the cleavage reaction necessary toseparate the fusion tag from a target protein have inherent difficultiesmanifesting themselves as: (i) non-specific proteolytic attack of thetarget protein; (ii) the need for elevated temperatures for efficientcleavage, often resulting in the denaturation or aggregation of thetarget protein; (iii) incomplete proteolytic processing resulting inpartially cleaved target protein, thereby significantly reducing theyield and/or introducing heterogeneity to the purified protein; (iv)additional purification steps are necessary to separate the cleavedtarget protein from the fusion tag, deactivate and remove the processingprotease and exchange or desalt buffer components.

Several fusion protein technologies are known and may be used, such asby use of:

-   Glutathione-S-Transferase GST (S. Markrides, Micro. Biol. Rev.,    60 (1996) 512-538; J. Nilsson, S. Ståhl, J. Lundeberg, M. Uhlén and    P.-Å. Nygren, Prot. Expr. Purif., 11 (1997) 1-16);-   polyhistidine tags [E. Hochuli, W. Bannwarth, H. Döbeli, R. Gentz    and D. Stüber, Bio/Technology, 6 (1988) 1321-1325; E. Hochuli, H.    Döbeli and A. Schacher, J. Chromatogr., 411 (1987) 177-184);-   FLAG-tags [B. L. Brizzard, R. G. Chubet, D. L. Vizzard,    BioTechinques, 16 (1994) 730-734; T. P. Hopp, K. S. Prickett, V. L.    Price, R. T. Libby, C. J. March, D. P. Cerretti, D. L. Urdal, and-   P. J. Conlon, Bio/Technology, 6 (1988) 1204-1210; A. Knappik and A.    Plückthun, BioTechniques, 17 (1994) 747-761];-   thioredoxin [E. R. LaVallie, E. A. DiBlasio, S. Kovacic, K. L.    Grant, P. F. Schendel and J. M. McCoy, Bio/Technology, 11 (1993)    187-193; Z. Lu, E. A. DiBlasio-Smith, K. L. Grant, N. W.    Warne, E. R. LaVallie, L. A. Collins-Racie, M. T. Follettie, M. J.    Williams and J. M. McCoy, J. Biol. Chem., 271 (1996)    5059-5065; D. L. Wilkinson, N. T. Ma, C. Haught and R. G. Harrison,    Biotechnol. Prog., 11 (1995) 265-269.];-   Protein A [J. Nilsson, P. Nilsson, Y. Williams, L. Pettersson, M.    Uhlén and P.-Å. Nygren, Eur. J. Biochem, 224 (1994) 103-108; E.    Samuelsson, T. Moks, B. Nilsson and M. Uhlén, Biochemistry,    33 (1994) 4207-4211; M. Uhlén, B. Nilsson, B. Guss, M. Lindberg, S.    Gatenbeck and L. Philipson, Gene, 23 (1983) 369-378.];-   Strep-tag [J. Nilsson, M. Larsson, S. Ståhl, P.-Å. Nygren and M.    Uhlén M. J. Mol. Recognit., 9 (1996) 585-594; T. G. M. Schmidt    and A. Skerra, Prot. Eng., 6 (1993) 109-122.]; and-   Maltose-binding protein [H. Bedouelle and P. Duplay, Eur. J.    Biochem., 171 (1988) 541-549; C. di Guan, P. Li, P. D. Riggs and H.    Inouye, Gene, 67 (1988) 21-30; C. V. Maina, P. D. Riggs, A. G.    Grandea III, B. E. Slatko, L. S. Moran, J. A. Tagliamonte, L. A.    McReynolds and C. diGuan, Gene, 74 (1988) 365-373].    Glutathione-S-Transferase (GST) Fusions

Currently available Glutathione-S-Transferase (GST) systems can be usedwith GST-affinity resins using coupled glutathione or glutathionederivatives as ligand and GST or GST derivatives for binding as afusion-protein binding component.

Polylhistidine Tags

Currently available polyhistidine (or derivatives, e.g. His-Glu-His-Glusystems included) can be used in association with a number of types ofimmobilizing chelating resins, such as IMAC-chelating (ex AmershamBiosciences of Uppsala, Sweden), Ni-NTA (ex Qiagen), Talon (exClonetech), Tentatcle (ex Merck) etc.

Maltose Binding Protein (MBP) Fusions

Currently available MBP systems can be used, with MBP-affinity resinsusing coupled amylose (or derivatives, or other compatible sugars) andMBP (or derivatives) for binding as a fusion-protein binding component.

Immobilization of the target protein or polypeptide on the solid phasealso may be achieved by use of currently available or custom designedaffinity purification systems based on immuno-recognition, either byantibodies or antibody/antigen fragments bonded to suitable resins. Theimmobilization could be mediated by a specific and/or non-specificbinder (molecule and/or moiety with some affinity for the targetprotein).

Still other means for immobilizing the protein or polypeptide may beused, provided they selectively bind the protein or polypeptide to bemodified, possibly associated to a moiety capable of selectiveimmobilization on the solid phase. Such may be a moiety attachedchemically to the protein or polypeptide, e.g. by covalent binding,ionic binding etc, or may be a moiety such as a fusion tag. Thisimmobilization moiety should bind sufficiently strongly both to theprotein or polypeptide and the solid phase, to allow the protein orpolypeptide to remain imrnobilized during the modifying reaction, butstill in a reversible way to allow for subsequently collecting themodified protein or polypeptide, by breaking the bond between either theimmobilization moiety and the solid phase or between protein orpolypeptide and the immobilization moiety, or both. Suitable currentlyavailable examples are Heparin, Biotin/Streptavidin, nucleotide bindingsolid phases, as well as ion exchange resin.

Heparin Sepharose is an affinity chromatography resin. A HeparinSepharose resin provides fast, preparative separations of proteins andother biomolecules based on their affinity for heparin. Heparin is anaturally occurring glycosaminoglycan, which is an effective affinitybinding and cation ion exchange ligand for a wide range of biomolecules,including DNA binding proteins, coagulation factors and other plasmaproteins, lipoproteins, protein synthesis factors, enzymes that act onnucleic acids and steroid receptors. By coupling heparin to Sepharose™with a chemically optimized linkage, an excellent medium for affinitypurification is provided.

Purified Streptavidin isolated from Streptomyces avidinii is immobilizedon Sepharose™ beads. The immobilized streptavidin binds biotin andbiotinylated substances and can be used for affinity chromatographyapplications. The interaction between streptavidin and biotin is verystrong. It can be used in the purification of antigens, wherebiotinylated antibodies are incubated with antigen. The biotinylatedantibody-antigen complex binds to HiTrap Streptavidin from which theantigen can be eluted. Another example is to utilize the interactionbetween 2-iminobiotin and streptavidin, eluting the bound substances atpH 4.

Nucleotide binding may be performed on Blue Sepharose 6, which isCibacron Blue 3G coupled to Sepharose. A member of the BioProcess™ Mediafamily. It is particularly suitable for the isolation and purificationof albumin, interferon, a broad range of nucleotide requiring enzymes,α-macro-globulin, coagulation factors, and nucleic acid bindingproteins.

Ion exchange (IEX) separates proteins with differences in charge to givea very high resolution separation with high sample loading capacity. Theseparation is based on the reversible interaction between a chargedprotein and an oppositely charged chromatographic medium. Proteins areimmobilized, as they are loaded onto a column. Conditions are thenaltered so that the immobilized substances are eluted differentially.This elution is usually performed by increases in salt concentration orchanges in pH. Changes are made stepwise or with a continuous gradient.Most commonly, samples are eluted with salt (NaCl), using a gradientelution. Target proteins are concentrated during binding and collectedin a purified, concentrated form.

Hydrophobic interaction separates proteins with differences inhydrophobicity. The separation is based on the reversible interactionbetween a protein and the hydrophobic surface of a chromatographicmedium. High ionic strength buffer enhances these interactions. Samplesin high ionic strength solution (e.g. 1.5 M ammonium sulphate) areimmobilized, as they are loaded onto a column. Conditions are thenaltered so that the immobilized substances are eluted differentially.Elution is usually performed by decreases in salt concentration. Changesare made stepwise or with a continuous decreasing salt gradient. Targetsare concentrated during immobilization and collected in a purified,concentrated form. Other elution procedures include reducing eluentpolarity (ethylene glycol gradient up to 50%), adding chaotropic species(urea, guanidine hydrochloride) or detergents, changing pH ortemperature.

Reverse Phase chromatography separates proteins and peptides withdiffering hydrophobicity based on their reversible interaction with thehydrophobic surface of a chromatographic medium. Samples areimmobilized, as they are loaded onto a column. Conditions are thenaltered so that the immobilized substances are eluted differentially.Due to the nature of the reversed phase matrices, immobilization isusually very strong and requires the use of organic solvents and otheradditives (ion pairing agents) for elution. Elution is usually performedby increases in organic solvent concentration, most commonlyacetonitrile: Samples, which are concentrated during the binding andseparation process, are collected in a purified, concentrated form.

According to one aspect of the invention, kits are provided. Kitsaccording to the invention may include a number of suitable components,depending on the starting material, the desired modification(s), thepurpose of the modified protein or polypeptide etc.

A kit according to the invention comprises components for modifying animmobilized protein or polypeptide, i.e. at least one chemicallyreactive system or at least one enzymatically reactive system, or acombination of both systems.

In relation to drug discovery and disease detection, profiling andtreatment, a kit for detection of a post-translationally modifiedprotein or polypeptide may comprise all the components for production ofthe post-translationally modified protein or polypeptide, or thepost-translationally modified protein or polypeptide per se, andcomponents necessary for raising antibodies against the modified proteinor polypeptide. In addition, this applies to diagnostic assays andscreens that may be used as analytical tools or in high-throughputscreening applications.

A chemically reactive system, may comprise any number of reactivecomponents suitable for performing a given modification reaction, e.g.one or several reagents as well as catalysts. The chemicals may have anysuitable form, such as powder, liquid solution, emulsion, suspensionetc.

A reactive system of the invention may include biological components tocomplete the reaction, such as biological membranes; isolated andreconstituted enzymes and cellular components (including (endogenous orsupplemented) lipids, co-factors, salts, substrates).

Bioactive membranes and/or programmed lysates applicable in the methodand kits according to the invention can be endogenous biologicalmembranes or cellular solutions extracted from an organism, collectedand used in an alternative system. These membranes and/or lysates arebiologically active and perform as a function of their components, theirorganization, their stoichiometry, and primarily, their inherentfunction in their endogenous environment (a membrane or mulitmericcomplexes). Additional factors include, unknown biologically relevantcomponents that have not been identified or characterized. The isolationof these bioactive membranes and/or programmed lysates can alsoencompass the reconstitution of the active form of the membrane and/orcellular components by adding the active components and substances thatprovide the bioactive membrane and/or lysate with its active function.

The enzymatically reactive system comprises at least one enzyme capableof catalyzing a modification reaction and any auxiliary componentsnecessary for the modification reaction to occur. The enzyme may be inany form suitable for the purpose of the invention. E.g. it may be apurified preparation to be stored at a temperature between −20° C. and−80° C., or a preparation in a suitable buffer, to be stored at atemperature preferably of about +4° C. The enzyme preparation may alsobe in a lyophilized form.

A kit according to the invention may also comprise components forimmobilizing the protein or polypeptide on a solid phase.

Furthermore, a kit according to the invention may comprise componentsfor freeing and collecting the modified protein or polypeptide from thesolid phase.

In one embodiment, in addition to the components for modifying animmobilized protein or polypeptide, a kit according to the inventioncomprises: components for producing/obtaining a protein or polypeptideto be modified; components for immobilizing the protein or polypeptideon a solid phase; components for freeing and collecting the modifiedprotein or polypeptide from the solid phase; and components fortesting/operating/monitoring the process.

The components for obtaining a protein or polypeptide to be modified maycomprise e.g. a vector coding for the protein, optionally an affinityfusion tag construct; a bacterial strain suitable for overexpressing theprotein or polypeptide; reagents and substrates suitable for cultivatingthe bacteria and enhance overexpression.

The vector coding for a protein e.g. may be a plasmid vector, such as apGEX vector or derivation thereof.

The bacterial strain suitable for overexpressing the protein may be e.g.BL21 of Eschierichia coli or a derivation thereof.

The reagents and substrates suitable for cultivating the bacteria andenhance overexpression may be e.g. growth media such as Luria Broth,inducing reagent (IPTG), antibiotics for antibiotic resistance andprotease inhibitors.

The components for immobilizing the protein on a solid phase maycomprise a suitable solid phase and a liquid phase comprising protein orpolypeptide purification reagents.

The solid phase may be any solid phase capable of immobilizingselectively the protein or polypeptide of interest, e.g. affinity resinsor ion exchange gels. Preferably the solid phase is an affinity resin,e.g. Glutathione Sepharose, HiTrap™ IMAC chelating, from AmershamBiosciences, or derivatives thereof.

The protein purification reagents may be e.g. buffers, such as PBS;reducing agent, such as DTT; elution competitors, such as reducedglutathione or imidazole; protease inhibitors; and detergents.

The components for modifying the immobilized protein may comprise one orseveral modifying enzymes, selected in accordance with the particularmodification(s) one desire to perform, as well as any auxiliarycomponents required or suitable for performing the modificationreaction(s).

In the case of a kit comprising an enzymatic modification system, thechoice of the enzyme of course is primordial for effecting the desiredmodification. It is within the knowledge of the person skilled in theart to select a proper enzyme for any particular modification reaction.E.g. to perform a phosphorylation, a Icinase is required, whereas toperform a glycosylation, a glycosylase is required. In the case of a kitcomprising several enzymes, these may be provided separately or as amixture, or as a combination of both.

For selection of suitable enzymatic modification components, referencemay be made to web sites such as:

EXPASY-Post-Translational Modification Predictions

Swiss Institute of Bioinformatics (SIB) Appel R. D., Bairoch A.,Hochstrasser D. F. A new generation of information retrieval tools forbiologists: the example of the ExPASy WWW server. Trends Biochem. Sci.19:258-260(1994). (http://www.expasy.ch/tools/#ptm).

PhosphoBase

Kreegipuu A, Blom N, Brunak S (1999), “PhosphoBase, a database ofphosphorylation sites: release 2.0.”, Nucleic Acids Res 27(1):237-239(http://www.cbs.dtu.dk/databases/PhosphoBase/).

O-GLYCBASE version 4.0:

Database of O-glycosylated proteins. Ramneek Gupta, Hanne Birch,Kristoffer Rapacki, Søren Brunak and Jan E. Hansen. Nucleic AcidsResearch, 27: 370-372, 1999.(http://www.cbs.dtu.dk/databases/OGLYCBASE/).

Protein Information Resource

A Division of National Biomedical Research Foundation ProteinInformation Resource, National Biomedical Research Foundation 3900Reservoir Rd., NW, Washington, D.C. 20007, USA(http://pir.georgetown.edu/).

The auxiliary components required or suitable for performing themodification reaction(s) may comprise buffers, such as Tris-HCl;detergents; salts; reducing agents; required substrates/co-factors, e.g.ATP, phosphatidylserine; and protease inhibitors.

Herein below, a list of components, necessary (n) and optional (O), ofsome kits according to the invention, is provided:

-   Phosphorylation: kinase (n), buffers (O), substrates/co-factors    (ATP; n), additives (O), salts (O);-   Dephosphorylation: phosphatase (n), buffers (O),    substrates/co-factors (ATP; n), additives (O), salts (O);-   SUMOylation: carboxy-terminal hydrolase (O), activating enzyme (n),    conjugating enzyme (n), ligating enzyme (n), buffers (O),    substrates/co-factors (ATP; n), additives (O), salts (O);-   Ubiquitinylation: carboxy-terminal hydrolase (O), activating enzyme    (n), conjugating enzyme (n), ligating enzyme (n), buffers (O),    substrates/co-factors (ATP; n), additives (O), salts (O);    Carboxymethylation: carboxymethylase (n), buffers (O),    substrates/co-factors (ATP; n), additives (O), salts (O);-   Acetylation: acetyltransferase (n), buffers (O),    substrates/co-factors (ATP; n), additives (O), salts (O);-   Deacetylation: deacetyltransferase (n), buffers (O),    substrates/co-factors (ATP; n), additives (O), salts (O);-   Amidation: acyltransferase/amidase (n), buffers (O),    substrates/co-factors (ATP; n), additives (O), salts (O);-   Methylation: methyltransferase (n), buffers (O),    substrates/co-factors (ATP; n), additives (O), salts (O);-   Demethylation: carboxy-methyltransferase (n), buffers (O),    substrates/co-factors (ATP; n), additives (O), salts (O);-   Carboxylation: carboxylase (n), buffers (O), substrates/co-factors    (ATP; n), additives (O), salts (O);-   Carboxyamylation: carboxylase (n), buffers (O),    substrates/co-factors (ATP; n), additives (O), salts (O);-   Sulphation: sulphotransferases (n), buffers (O),    substrates/co-factors (ATP; n), additives (O), salts (O);-   Hydroxylation: hydroxylase (n), buffers (O), substrates/co-factors    (ATP; n), additives (O), salts (O);-   ADP-ribosylation: ADP-ribosylation factors (ARFs; n), buffers (O),    substrates/co-factors (ATP; n), additives (O), salts (O);-   Maturation: proteolytic processing proteases (n), buffers (O),    substrates/co-factors (ATP; n), additives (O), salts (O);-   Adenylation: adenylyl transferase (n), buffers (O),    substrates/co-factors (ATP; n), additives (O), salts (O);-   Glycosylation: glycosylase (n), buffers (O), substrates/co-factors    (ATP; n), additives (O), salts (O); and-   Prenylation: prenyltransferase (n), buffers (O),    substrates/co-factors (ATP; n), additives (O), salts (O).

The components for freeing and collecting the modified protein from thesolid phase may comprise proteolytic processing enzyme, such asPreScission™ protease or thrombin; cleavage reagents, such as buffers,e.g. Tris; salts; detergents; reducing agents and, if required, affinityresins to capture protease, e.g. HiTrap benzamidine column (sold byAmersham Biosciences), or derivatives thereof.

The components for testing/operating/monitoring the process may comprisehardware, such as a recirculation unit, e.g. ÄKTA platform recirculationconnector kit; application notes/technical support, reagents forperforming control reactions, e.g. to test fusion tag, such asGST-fusion or polyhistidine fusion that can be immobilized, modified andcleaved easily.

Some examples of kits are given herein below:

Kit for Phosphorylation of GST-Fusion Protein or Polyeptide

Such kit for example may contain any/all (or other) components:

-   (i) plasmid vector coding for affinity fusion tag construct (e.g.    pGEX vector or derivation thereof);-   (ii) bacterial strain (e.g. BL21, or derivation thereof);-   (iii) affinity resin (e.g. Glutathione Sepharose (GSTrap) or    derivatives thereof);-   (iv) protein expression reagents (e.g. growth media (LB), inducing    reagent (IPTG), antibiotics for antibiotic resistance, protease    inhibitors)*;-   (v) protein purification reagents (e.g. buffers (PBS), reducing    agents (DTT), elution competitor (reduced glutathione), protease    inhibitors, detergents);-   (vi) modifying enzyme(s) (e.g. specific kinase (Protein Kinase C;    PKC, or other kinase(s), or a mixture of known kinases, e.g.    PKC+MAPK+casein kinase II+ . . . + . . . + . . . ) to allow for    screening of potential phosphorylation sites on a systematic and    controlled manner; programmed lysate may also be used (such as    eukaryotic cell lysate that is capable of making the desired    modification reaction));-   (vii) master mix of modifying reaction components (e.g. 10× buffers    (Tris-HCl), detergents, salts, reducing agents, required    substrates/co-factors (ATP, phosphatidyl-serine), protease    inhibitors);-   (viii) proteolytic processing enzyme (e.g. PreScission protease);-   (ix) cleavage reagents (e.g. 10× buffers (Tris), salts, detergents,    reducing agents);-   (x) available hardware upgrades (e.g. ÄKTA platform recirculation    connector kit);-   (xi) application note/technical support (e.g. working methods on    available platforms, i.e., an off the shelf method to work for    GST-fusion proteins on an ÄKTA platform in an automated manner);-   (xii) control reactions (e.g. test GST-fusion that can be    immobilized, modified and cleaved easily)*.    *optional components.    Kit for Phosphorylation of Polyhistidine Tagged Fusion Protein or    Polypeptide

Such kit for example can contain any/all (or other) components:

-   i) plasmid vector coding for affinity fusion tag construct (e.g. his    tagged vector or derivation thereof);-   (ii) bacterial strain (e.g. BL21, or derivation thereof);-   (iii) affinity resin (e.g. HiTrap IMAC chelating, or derivatives    thereof);-   (iv) protein expression reagents (e.g. growth media (LB), inducing    reagent (IPTG), antibiotics for antibiotic resistance, protease    inhibitors)*;-   (v) protein purification reagents (e.g. buffers (PBS), reducing    agents (DTT), elution competitor (imidazole), protease inhibitors,    detergents);-   (vi) modifying enzyme(s) (e.g. specific kinase (Protein Kinase C;    PKC, or other kinase(s), or a mixture of known kinases, e.g.    PKC+MAPK+casein kinase II+ . . . + . . . + . . . ) to allow for    screening of potential phosphorylation sites on a systematic and    controlled manner; programmed lysate may also be used (such as    eukaryotic cell lysate that is capable of making the desired    modification reaction));-   (vii) master mix of modifying reaction components (e.g. 10× buffers    (Tris-HCl), detergents, salts, reducing agents, required    substrates/co-factors (ATP, phosphatidylserine), protease    inhibitors);-   (viii) proteolytic processing enzyme (e.g. Thrombin);-   (ix) cleavage reagents (e.g. 10× buffers (Tris), salts, detergents,    reducing agents);-   (x) affinity resin to capture protease (e.g. HiTrap benzamidine    column, or derivatives thereof);-   (xi) available hardware upgrades (e.g. ÄKTA platform recirculation    connector kit);-   (xii) application note/technical support (e.g. working methods on    available platforms, i.e., an off the shelf method to work for    GST-fusion proteins on an ÄKTA platform in an automated manner);-   (xiii) control reactions (e.g. test polyhistidine fusion that can be    immobilized, modified and cleaved easily)*.    *optional components.    Kit for Glycosylation of GST-Fusion Protein or Polypeptide

Such kit for example can contain any/all (or other) components:

-   (i) plasmid vector coding for affinity fusion tag construct (e.g.    pGEX vector or derivation thereof);-   (ii) bacterial strain (e.g. BL21, or derivation thereof);-   (iii) affinity resin (e.g. Glutathione Sepharose (GSTrap) or    derivatives thereof);-   (iv) protein expression reagents (e.g. growth media (LB), inducing    reagent (IPTG), antibiotics for antibiotic resistance, protease    inhibitors)*;-   (v) protein purification reagents (e.g. buffers (PBS), reducing    agents (DTT), elution competitor (reduced glutathione), protease    inhibitors, detergents);-   (vi) stable “programmed” lysates (e.g. microsomal system with all    modifying enzymes (glycosylases) contained in biomembranes, or in    solution, that can be incubated with the immobilized target protein    modifying the target protein in a recirculated manner in-line,    on-column in an automated manner; purified (possibly recombinant)    components reconstituted in reaction mix may also be used);-   (vii) master mix of modifying reaction components (e.g. 10× buffers    (Tris-HCl), detergents, salts, reducing agents, required    substrates/co-factors (ATP, phosphatidylserine), protease    inhibitors);-   (viii) proteolytic processing enzyme (e.g. PreScission protease);-   (ix) cleavage reagents (e.g. 10× buffers (Tris), salts, detergents,    reducing agents);-   (x) available hardware upgrades (e.g. ÄKTA platform recirculation    connector kit);-   (xi) application note/technical support (e.g. working methods on    available platforms, i.e., an off the shelf method to work for    GST-fusion proteins on an ÄKTA platform in an automated manner);-   (xii) control reactions (e.g. test GST-fusion that can be    immobilized, modified and cleaved easily)*.    *optional components.    Kit for Amidation of GST-Fusion Peptide (C-Terminal Modification)

Such kit for example can contain any/all (or other) components:

-   (i) plasmid vector coding for affinity fusion tag construct (e.g.    pGEX vector or derivation thereof);-   (ii) bacterial strain (e.g. BL21, or derivation thereof);-   (iii) affinity resin (e.g. Glutathione Sepharose (GSTrap) or    derivatives thereof);-   (iv) protein expression reagents (e.g. growth media (LB), inducing    reagent (IPTG), antibiotics for antibiotic resistance, protease    inhibitors)*;-   (v) protein purification reagents (e.g. buffers (PBS), reducing    agents (DTT), elution competitor (reduced glutathione), protease    inhibitors, detergents);-   (vi) modifying enzyme(s) (e.g. peptidylglycine alpha-amidating    monooxygenase (PAM); programmed lysate (such as eukaryotic cell    lysate that is capable of making the desired modification reaction)    as well as chemical modification methods may also be used);-   (vii) master mix of modifying reaction components (e.g. 10× buffers    (Tris-HCl), detergents, salts, reducing agents, required    substrates/co-factors (ATP, phosphatidylserine), protease    inhibitors);-   (viii) proteolytic processing enzyme (e.g. PreScission protease);-   (ix) cleavage reagents (e.g. 10× buffers (Tris), salts, detergents,    reducing agents);-   (x) available hardware upgrades (e.g. ÄKTA platform recirculation    connector kit);-   (xi) application note/technical support (e.g. working methods on    available platforms, i.e., an off the shelf method to work for    GST-fusion proteins on an ÄKTA platform in an automated manner);-   (xii) control reactions (e.g. test GST-fusion that can be    immobilized, modified and cleaved easily)*.    *optional components.    Kit for Detection of a Post-Translationally Modified Protein or    Polypeptide (PTM Biomolecule)

Such kit for example can contain any/all (or other) components:

-   (i) PTM biomolecule (ii) antibodies raised specifically against PTM    biomolecule (in negative background on unmodified biomolecule)-   (iii) master mix of antibody reaction components (e.g. 10× buffers    (Tris-HCl), detergents, salts, reducing agents, required    substrates/co-factors, protease inhibitors);-   (iv) western blotting or ELISA detection systems-   (v) available hardware upgrades-   (vi) application note/technical support (e.g. working methods on    available platforms, i.e., an off the shelf method to work for    detection of PTM identified proteins on an existing platform in an    automated manner);-   (xii) control reactions (e.g. test PTM biomolecules with antibodies    that can be detected and identified easily)*.    *optional components.

The advantage provided by the various aspects of the invention resortfrom the above disclosure. The processes of the current state-of-the-artin producing modified proteins/peptides, have many drawbacks includinginefficiency of production, expense, poor levels of purity, poor yield,extreme difficulties in isolation of natural product and heterogeneity.By the method of the invention, a systematic controlled approach isprovided instead of relying solely on serendipitous interactions(programmed lysates, incubating target protein to be modified witheukaryotic lysates which contain endogenous modifying proteins and hopefor target protein modifications after prolonged incubation periods) ormultiple processing steps (in vitro purification of all the necessarycomponents then reconstitute the entire mix to create the desiredmodification (low level) only to have to purify away all the reactioncomponents in order to isolate the modified target protein). Theintegration of several pre-existing methods coupled to applicationsinvolving the systematic control of experimental design and conditionsin an automated manner is entirely unique and novel. The currentstate-of-the-art is moving in the opposite direction towards biologicalmodifications using selected eukaryotic cell lines where the finalproduct is under the entire control of the organism. This often resultsin low levels of modifications, heterogeneous modifications, spuriousmodifications and the experimenter starts processing the product withcompromised levels of material both in yield and quality. In contrast tothis, by the method of the invention, the reactive system is driven andcontrolled entirely in a comprehensive systematic way that can beautomated and can produce material that is simply not obtainable inreasonable yields in any currently existing system.

EXAMPLES

To further elucidate the invention, two examples of post-translationalmodification of a protein by a method according to the invention aredescribed herein below as Examples 1 and 2.

Further examples, i.e. Examples 3 and 4 respectively generallyillustrate the scale-up of process steps of the inventive method. Inthese examples, the immobilized protein, which is a fusion protein, istreated with Pre-Scission protease to cleave off the fusion partner.However, it should be understood that any enzyme, supplemented withsuitable components necessary for the reaction to occur, such asco-factors and substrates, could be used in place of the protease,according to the PTM which it is desired to perform.

In addition, Examples 3 and 4 illustrate the processing of a difficulttarget (TDG), making the application to novel experimentally challengingtargets more feasible.

Example 1

In the present example a human eukaryotic cell receptor protein,recombinantly produced in a bacterial host as a fusion protein, wasphosphorylated by a method according to the invention.

Methodological and Experimental Summary

Immobilization, Purification, Modification and On-Column Cleavage of aReceptor Protein

An over-expressed GST:: fusion protein is: affinity bound andimmobilized on Glutathione Sepharose resin; post-translationallymodified in-line on-column by phosphorylation reaction with PKC and/orMAPK enzymes; efficiently cleaved on-column and eluted as a homogenousmodified product. The purity of the eluted proteins is evaluated bySDS-PAGE and mass spectrometry.

After selecting for conditions which produce high levels of expressedGST::fusion protein, the GST::fusion protein containing lysate isclarified by subsequent centrifugation steps at ˜70,000×g and at˜300,000×g. Following the ultracentrifugation step, the clarified lysatecontaining GST::fusion protein is loaded on a GSTrap™ FF column with PBSbinding buffer and the unbound fraction passes through the column. PBSbinding buffer is used to wash the GSTrap™ FF column until theabsorbance baseline returns. At this point the buffer is exchanged toPhosphorylation buffer (PKC-pb or MAPK-pb) and equilibrated. Theimmobilized GST::fusion protein is then incubated with modifying enzyme(PKC and/or MAPK) injected and applied to the column in iterative steps.Single specific modifications, or multiple modifications are possible.The primary requisite is buffer exchange to optimum reaction buffers(PKC-pb and MAPK-pb) for each modification reaction. Following themodification reactions, the immobilized GST::fusion protein is washed bybuffer exchange to cleavage buffer. The cleavage buffer acts primarilyto equilibrate the GSTrap™ FF column prior to proteolytic cleavage byPreScission™ protease of the bound GST::fusion protein. This finalbuffer exchange plays an important role in downstream processessensitive to phosphate salts, such as crystallization conditionscreening, and metal dependent biochemical assays. After the absorbancebaseline returns, PreScission™ protease is loaded on the GSTrap™ FFcolumn and the system is in a ‘closed’ position incubating for ˜12 h.Following incubation, the cleaved modified protein is eluted. After theabsorbance baseline has returned, a 100% step gradient of reducedglutathione containing buffer, elution buffer, is introduced and acts asa competitor for GST binding sites. The bound GST::linker (proteolyticcleavage reaction product) and PreScission™ protease elute from thecolumn. The column is regenerated after the absorbance baseline hasreturned and can be re-equilibrated with binding buffer for subsequentpurification and on-column cleavage runs. In this example, this methodcan be scaled from small-scale (<1 ml cultures) to larger-scale (>20litre cultures) in a linear manner and is directly proportional tomaterials and yield reproducing consistent levels of protein production.The overall purification, immobilization, modification and on-columncleavage strategy produces highly pure, enriched modified protein with ayield of ˜0.8 mg/litre culture in the purity range of 95% pure proteincompleted in a single chromatographic step.

GST::Fusion Protein Binding

The supernatant containing the GST::fusion protein fraction from theultracentrifugation step (soluble or membrane containing fractions) wasloaded on a GSTrap™ FF column (5 ml; Amersham Biosciences, Uppsala,Sweden), pre-equilibrated with PBS as binding buffer, at a flow rate of1 ml/min. For all chromatographic steps, an ÄKTA™ explorer (AmershamBiosciences, Uppsala, Sweden) was used enclosed in a refrigeration unitcooled to 4° C. to ensure protein stability and reduce proteindegradation. Chromatographic profiles monitor continuously absorbance(260 and 280 nm) and conductivity (mS/cm). The bound material was washedwith PBS buffer until the absorbance baseline had returned. Once thebaseline was stable, the buffer was exchanged with eitherPKC-Phosphorylation buffer (PCK-pb; 20 mM HEPES-KOH at pH 7.4; 10 mMMgCl₂; 1.7 mM CaCl₂; 600 μg/ml phosphatidyl serine; 1 mM DTT and 50 μMATP) or MAPK-Phosphorylation buffer (MAPK-pb; 50 mM Tris-HCl at pH 8.0;0.5 mM EDTA; 25 mM MgCl₂; 1 mM DTT; 50 μM ATP and 10% glycerol).Phosphorylation buffer equilibration was continued until stableabsorbance baseline was achieved. At this stage, buffer flow wasarrested.

In Vitro Phosphorylation of GST::Fusion Protein

For the in vitro phosphorylation reaction of immobilized GST::fusionprotein (bound on the GSTrap column) with PKC and/or MAPK, the kinasewas injected and incubated in-line on-column with 25 ng of PKC in PKC-pbor 25 ng of MAPK in MAPK-pb. All phosphorylation reactions were carriedout at 4° C. for 30 minutes-12 hours, in the presence of protease andphosphatase inhibitors (1 μM okadaic acid; 200 μM Na₃VO₄). For iterativemodification reactions, the immobilized fusion protein was washed withthe subsequent buffer and reaction components prior to injection andincubation with modifying enzyme. This step can be cycled until thedesired modifications using defined enzymes reach completion. Theaddition of the modifying enzyme in appropriate buffer component systemcan be applied to the column in a tightly controlled and monitoredmanner using a closed recirculation loop (P-950 pump connected tosample/super loop). This recirculating modifying enzyme method allowsfor multiple reaction rounds, decreases overall reaction time, decreasesmodifying enzyme concentrations necessary to complete the reaction andincreases the population of modified target protein.

On-Column Cleavage Reaction

This method utilizes the technologies of a GST::fusion protein linkedwith an infrequently biologically occurring proteolytic cleavage site.In conjunction with glutathione affinity columns and a highly specificengineered protease to purify desired target proteins in high yieldswith high levels of enrichment. PreScission protease (AmershamBiosciences, Uppsala, Sweden) is a genetically engineered fusion proteinconsisting of GST fused to a modified human rhinovirus 3C protease.PreScission protease (2 Units enzyme/100 μg of bound fusion GST::fusionprotein) was diluted in Cleavage buffer equal to 90% of the volume ofthe GSTrap™ FF column and injected into the column at an increased flowrate of ˜5-7 ml/min. Following injection, the column was placed in aclosed flow status and the system was incubated on-line for 12-16 h at4° C.

Elution of ‘Native’ Protein

An auxiliary GSTrap™ FF column (1 ml) pre-equilibrated with Cleavagebuffer was connected downstream of the primary cleavage reaction columnin-line with the fraction collector. Cleaved modified target proteinelution occurs immediately upon flow start-up with Cleavage buffer at ˜1mmin. Following cleaved target protein elution and the return of theabsorbance baseline, the GST-affinity peak was eluted with Elutionbuffer (50 mM Tris-HCl at pH 8.0 and 10 mM reduced glutathione) in afull step gradient (100% elution buffer).

Column Regeneration/Equilibration

Following the elution of the target protein and the GST-affinityproteins, the column can be equilibrated for subsequent purificationruns. Column equilibration is completed by flushing the column with 3column volumes of Milli-Q water followed by 3 column volumes of PBSBinding buffer. This regeneration stage is important for the throughputof the protein production process and allows for multiple runs to becompleted in series.

Mass Spectrometry

Purified fusion protein was phosphorylated by PKC (Promega) according tomanufactures instructions and subjected to SDS-PAGE. Samples wereprepared according to Shevchenko et al. (Andrej Shevchenko, IgorChemushevich, Matthias Wilm, and Matthias Mann, Methods in MolecularBiology, vol. 146: Protein and Peptide Analysis: New Mass SpectrometricApplications, Ed.: J. R. Chapman, Humana Press Inc., Totowa, N.J.).After tryptic digest of phosphorylated and control samples, peptideswere extracted and analyzed by matrix-assisted laserdesorption/ionization mass spectrometry using Voyager BiospectrometryWorkstation with Delayed Extraction Technology, PerSeptive Biosystems,Inc. Obtained data was analyzed using Moverz software (Proteometrics,LLC). The mass spectrometric analysis showed that GST:: fusion proteinhad been post-translationally modified by PKC at a specific position.

Example 2

In this example, a human developmental regulatory protein wasimmobilized and purified as a polyhistidine tagged fusion protein andsubmitted to two independent phosphorylation modifications.

The target Protein is 58 kDa from Homo sapiens and is designated p58 inthis study. Parental vector encodes for p58 gene product in pBluescriptvector isolated from cDNA library.

Over-expression vector sub-cloned into N-terminal His-tag pQE80(His_(6x)::p58).

Post-translational phosphorylation modifications of p58 with ProteinKinase C (PKC) and/or Casein Kinase 2 (CK-II).

Sub-Cloning

The cDNA encoding for p58 was excised from the pBluescript vector andligated into a pQE80 vector creating p58-Q80. The p58 sub-clone wastransformed into an E. coli cloning cell line MC1061. The plasmidconstruct was amplified and purified with DNA minipreps and p58-Q80plasmid was transformed into the over-expression E. coli cell line M15.

Culturing

Overnight cultures of p58-Q80 transformed M15 bacteria were made byinoculating 5 ml of LB-media supplemented with Carbenicillin (100 μg/ml)and Kanamycin (50 μg/ml) with a single colony selected from the LB-agarplates. For the over-expression trials, 100 μl of overnight cultureinoculated 100 ml LB-media supplemented with Carbenicillin (100 μg/ml)and Kanamycin (50 μg/ml). Over-expression trials controlled variablessuch as: temperature (30° C. and 37° C.); induction time (OD₆₀₀≈0.6 andOD₆₀₀≈1.0); induction concentration, IPTG(isopropyl-thio-β-D-galactoside) final concentrations (0.1 mM and 1.0mM); and growth time, where growth curves were recorded observing formaximum amount of soluble over-expressed His_(6x)::p58 target proteinlevels.

-   -   Over-expression assays grew overnight cultures at 18-37° C.,        containing antibiotics (100 μg/ml Carbenicillin) from fresh        transformants in LB-Miller or 2×YT Medium.    -   Inoculate large cultures with {fraction (1/1000)} inoculant from        overnight cultures. LB-Miller or 2×YT Medium containing        antibiotics (100 μg/ml Carbenicillin, 18-37° C.).    -   Grow the cells at 18-37° C., shaking at 244 rpm until        OD₆₀₀≈0.6-1.0.    -   Induce the protein synthesis with 0.1-1.0 mM IPTG (final        concentration) and let grow for 3-4 hours.    -   Harvest the cells in IL centrifuge cylinders and centrifuge at        5000×g at 4° C. for 30 minutes.    -   Wash the cell pellet and resuspended the cells in TB buffer (9.1        mM HEPES, 55 mM MnCl₂, 15 mM CaCl₂, 250 mM KCl adjusted to pH        6.7).    -   Cells are then pooled and centrifuged in centrifuge tubes, spun        at 4000×g for 5 minutes at 4° C.        Cell Lysis    -   Cells were lysed by resuspending the pellet in Lysis buffer (50        mM Tris-HCl pH 8.0, 100 mM NaCl and 2% glycerol, supplemented        with: 50 mg of Lysozyme, EDTA-free Complete Protease Inhibitor        Cocktail, DNase 1 and 10 mM MgCl₂) on ice. The volume is        approximately 10 ml for a 500 ml culture, 40 ml for a 2 l        culture and 120 ml for a 5 l culture.    -   Lysis is performed in sealed centrifuge tubes to allow for        proper mixing    -   After pellet is homogenously resuspended in solution, freeze        pellet in N₂ (1) until pellet is completely frozen, then rapidly        transfer the tube to 20° C. water bath. Gently shake the tube to        thaw the frozen lysate evenly. Repeat this freeze/thaw step        three times.    -   After cell lysate is homogenously thawed after the third        freeze/thaw step, centrifuge lysate at 180,000×g for 30 minutes        at 4° C.    -   Remove supernatant and place clarified lysate in        ultracentrifugation tubes. Centrifuge at 300,000×g for 90        minutes at 4° C.        Immobilization    -   The supernatant containing the His_(6x)::p58 protein fraction        from the ultracentrifugation step was loaded on a HiTrap™ IMAC        affinity column (5 ml; Amersham Biosciences, Uppsala, Sweden),        pre-equilibrated with Binding buffer (50 mM Tris-HCl pH 8.0, 100        mM NaCl and 2% glycerol), at a flow rate of 1 ml/min. For all        chromatographic steps, an ÄKTAT™ explorer (Amersham Biosciences,        Uppsala, Sweden) was used enclosed in a refiigeration unit        cooled to 4° C. to ensure protein stability and reduce protein        degradation. Chromatographic profiles monitor continuously        absorbance (260 and 280 nm) and conductivity (mS/cm).    -   Pre-equilibrate the IMAC affinity column (charged with Co²⁺)        with Binding buffer.    -   Remove the ultracentrifugation supernatant and apply it to the        pre-equilibrated affinity column.    -   p58 protein is immobilized on the affinity column    -   Immobilized p58 is gently washed with ˜10-20 column volumes of        Binding buffer.    -   Bound sample is stringently washed with ˜10-20 column volumes of        Wash buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 2% glycerol and        10 mM imidazole).        In-Line on-Column Phosphorylation with PKC    -   Phosphorylation reaction of immobilized p58 fusion protein        (bound on the Co2+ affinity column) with PKC.    -   The kinase was injected and incubated in-line on-column with 25        ng of PKC in PKC-Phosphorylation buffer (PCK-pb; 20 mM HEPES-KOH        at pH 7.4; 10 mM MgCl₂; 1.7 mM CaCl₂; 600 μg/ml phosphatidyl        serine; 1 mM DTT and 50 UM ATP)    -   All phosphorylation reactions were carried out at 4° C. for 30        minutes-12 hours, in the presence of protease and phosphatase        inhibitors (Complete Protease Inhibitor (Roche); 1 μM okadaic        acid; 200 μM Na₃VO₄).    -   The modifying enzyme in appropriate buffer component system is        applied to the colurnn in a tightly controlled and monitored        manner using a closed recirculation loop (P-950 pump connected        to sample/super loop).    -   This recirculating modifying enzyme method allows for multiple        reaction rounds, decreases overall reaction time, decreases        modifying enzyme concentrations necessary to complete the        reaction and increases the population of modified target        protein.        In-Line on-Columnn Phosphorylation with CK-II    -   Phosphorylation reaction of immobilized p58 fusion protein        (bound on the Co2+ affinity column) with CK-II.

The kinase was injected and incubated in-line on-column with 25 ng ofCK-II in CK-II Phosphorylation buffer (CK-II-pb; 25 mM Tris-HCl at pH7.4; 1 mM EDTA, 1 mM DTT, 200 mM NaCl, 2% glycerol, 10 mM MgCl₂; 1.7 mMCaCl₂; 600 μg/ml phosphatidyl serine; 1 mM DTT and 50 μM ATP).

-   -   All phosphorylation reactions were carried out at 4° C. for 30        minutes-12 hours, in the presence of protease and phosphatase        inhibitors (Complete Protease Inhibitor (Roche); 1 μM okadaic        acid; 200 μM Na₃VO₄).

The modifying enzyme in appropriate buffer component system is appliedto the column in a tightly controlled and monitored manner using aclosed recirculation loop (P-950 pump connected to sample/super loop).

-   -   This recirculating modifying enzyme method allows for multiple        reaction rounds, decreases overall reaction time, decreases        modifying enzyme concentrations necessary to complete the        reaction and increases the population of modified target        protein.        Modified Target Protein Elution    -   Immobilized modified p58 is eluted with ˜4-10 column volumes of        Elution buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 2% glycerol        and 200 mM imidazole).    -   The eluted p58-containing fraction is then concentrated (either        by centrifugal methods or a with a pressurized ultra-filtration        device).        Analysis

Several different methods of analysis of modified p58 were performed foreach immobilization, modification and purification process: sodiumdodecyl sulphate polyacrylamide amide gel electrophoresis (SDS-PAGE)denaturing gel; native PAGE; Western blotting using different antibodiesraised against His6x and p58; and mass spectrometry.

I. SDS-PAGE

The modified p58 protein purity and modification was analyzed bySDS-PAGE (3.5% stacking gel and 12% separation gel). Samples were mixedwith 2×Fling-and-Gregerson sample buffer (55 mM Tris-HCl pH 6.8, 2% SDS,7% Glycerol, 4% β-mercaptoethanol and 0.01% Bromophenol blue), boiledfor 7 minutes at 95° C. and separated electrophoretically on thedenaturing SDS-PAGE. The gel was run at 120V for approximately 1 hourand visualized with Coomassie Brilliant Blue staining.

II. Native-PAGE

Non-reducing native-PAGE was performed on modified p58 using the samebuffers as with the SDS-PAGE, but without the denaturing reagent SDS,reducing agent β-mercaptoethanol and without boiling the samples.Electrophoreses was run at 120V for 2 hours, in the same manner asSDS-PAGE and visualized with Coomassie Brilliant Blue staining.

III. Immunoblotting

Western blots were made according to two different protocols. One wasprepared by electro-blotting the protein onto nitrocellulose membranefor 1 hour at 100 V and blocking the membrane with 1-5% BSA prior toincubating with the primary antibody. The other protocol blocked themembrane with low-fat non-dairy milk (LFNDM). Several antibodies wereassayed; anti-p58 (dilution 1:2,000) directed against p58 andanti-RGS-His6_(x) (dilution (1:2,000) directed against RGS-His6_(x)sequences. For secondary antibodies, anti-mouse IgG (dilution 1:30,000)and goat anti-rabbit (dilution 1:4,000) were used.

Mass Spectrometry

-   -   Purified p58 was phosphorylated by PKC and/or CK-II and        subjected to SDS-PAGE. Samples were prepared according to        Schevchenko et al. 2000. After tryptic digest of phosphorylated        and control samples, peptides were extracted and analysed by        matrix-assisted laser desorption/ionization mass spectrometry        using Voyager Biospectrometry Workstation with Delayed        Extraction Technology (PerSeptive Biosystems, Inc). Obtained        data were analysed using Moverz software (Proteometrics, LLC).

Conclusions

The p58 protein was produced as over-expressed recombinant material andsubjected to tightly controlled and systematic immobilization,modification and purification process producing a biologically relevantphosphorylated species. Previously, using currently availabletechnologies, a comprehensive study of this modified p58 species wouldnot be possible at this level of purity and scale in an efficientmanner. The final product was highly enriched in specificallyphosphorylated p58 as defined by SDS-PAGE, Native-PAGE, Immunoblottingand Mass Spectrometry. Currently, the modified p58 protein is being usedfor functional and structural studies as well as in the drug discoveryprocess.

Example 3

Purification of the HNF4α LBD using On-Column Cleavage Strategy andGSTPrep 16/10 Column

Bacterial Growth and Protein Expression.

Rat HNF4α ligand binding domain (LBD, aminoacids 133-368) was expressedas a fusion protein with GST using pGEX-6p expression plasmid and BL21C+E. coli host. Four liters of LB medium supplemented with 5% sucrose,ampicilin (100 μg/ml) and IPTG (0.1 mM) were inoculated with overnightbacterial culture to 0.1 OD_(600nm). Bacteria were grown in 2.5 l Tunairflasks (Shelton Scientific, Shelton, Conn., USA) at 180 rpm, 18° C. for17 hours. Cells were collected by centrifugation (Beckman J L A 8.1000,20 min., 12,000 g) and washed in PBS buffer (140 mM NaCl, 2.7 mM KCl, 10mM Na₂HPO₄, 1.8 mM KH₂PO₄ adjusted to pH 7.4).

Lysis and Lysate Clearance.

Bacterial pellet was resuspended in PBS supplemented with 1 mg/mllysozyme, 10 U/ml DNase, 5 mM MgCl₂. After 30 minutes of incubation onice, bacteria were subjected to sonication (12 min total, pulse 1 sec:2sec=on:off) with constant stirring on ice. Bacterial lysate wasclarified by centrifugation at 50,000×g, 4° C. for 30 minutes (Beckman,Type 45 Ti) and subsequent ultracentrifugation at 180,000×g, 4° C. for 1hour (Beckman, Type 60 Ti).

Chromatography.

Clarified bacterial lysate (120 ml) was applied on the GSTPrep 16/10column by the sample pump in the direct load mode. Column wasextensively washed with PBS and buffer was changed for PreScissionProtease cleavage buffer. For on-column cleavage, the column wasdisconnected from the system, 7 ml (⅓ of the column volume) of thecleavage buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mMDTT) containing PreScission Protease (160 U) was injected on the columnand incubated at 10° C. overnight. Cleaved product was collected in 22ml of the cleavage buffer using GSTrap 1 ml auxiliary column downstreamof the GSPrep 16/10 to eliminate GST and PreScission Protease leakage.The uncleaved protein was eluted by GSH elution buffer (50 mM Tris-HClpH 8.0, 10 mM reduced glutathione) In FIG. 6, A corresponds to elutionwith PBS, PreScission Protease cleavage buffer; B corresponds to elutionwith the Glutathione elution buffer. Flow rate: 2 ml/min.

Protein sample from the on-column cleavage was injected on the ResourceQ 6 ml column preequilibrated in 20 mM Tris-Cl pH 8.0, 50 mM NaCl.Unbound protein was washed by 150 mM NaCl until reaching baseline andHNF4α LBD was collected by 400 mM NaCl step in small fraction volumes(FIG. 7). This procedure both partially purified and concentrated targetprotein for the last purification step. In FIG. 7, A corresponds toelution with 20 mM Tris-Cl pH 8.0 and B corresponds to elution with 20mM Tris-Cl pH 8.0, 1M NaCl. Flow rate: 3 ml/min.

Fractions from the Resource Q column with highest protein concentrationwere pooled (0.8 ml) and injected on Superdex 75 16/60 columnequilibrated in elution buffer (20 mM HEPES pH 7.4, 140 mM NaCl). Thesample was fractionated at flow rate 0.25 ml/min overnight withautomatic fractionation at 5 nAU level (FIG. 8). Target protein waseluted in the peak with apparent molecular weight 58.5 kDa, clearlyseparated from aggregated and/or complexed HNF4α LBD with bacterialchaperones. Molecular weight corresponds to LBD dimer (57.8 kDa) whichis in agreement with the dimerisation properties of HNF4.

In FIG. 8, the arrows indicate elution volumes of the standard proteinsfrom the Gel Filtration LMW Calibration Kit. Fractions analyzed bySDS-PAGE are indicated by capital letters.

HNF4 LBD was collected in 7 ml and concentrated using Microsep 10.000MWCO (Pall Filtron) in gel filtration elution buffer to final volume 400μl and protein concentration 12 mg/ml.

The protein samples from rat HNF4α LBD purification were analysed bySDS-PAGE. The results are represented in FIG. 9, wherein (A) correspondsto a bacterial lysate; (B) corresponds to the flow-through GSTPrep 16/10column, (C) corresponds to on-column cleaved product, (D) corresponds toglutathione elution, (E) corresponds to concentrated protein by 40% Bstep on Resource Q 6 ml column, (F) corresponds to 100% B wash fromResource Q 6 m] column, (G,H,I) fractions from HiLoad 16/60 Superdex 75prep column.

The purified rat HNF4α LBD was identified by MALDI-TOF MS. FIG. 10Arepresents the m/z spectrum of the peptides from tryptic digest. Peptidepeaks corresponding to expected signals from rat HNF4α LBD are labelledwith their molecular weight. M—matrix peaks.

FIG. 10B gives the full length sequence of the rat HNF4α protein. Inbold, target protein corresponding to lig and binding domain is shown.Peptides identified by MALDI-TOF MS are underlined.

Example 4

Purification of the Tymidine DNA Glycosylase (TDG) Using On-ColumnCleavage Strategy and GSTPrep 16/10 Column

Bacterial Growth and Protein Expression

Mouse TDG was expressed as a fusion protein with GST using pGEX-6pexpression plasmid and BL21 C+E. coli host. Eight liters of LB mediumsupplemented with ampicilin (100 μg/ml) were inoculated by 3% overnightbacterial culture. Bacteria were grown in 2 l E-flasks at 180 rpm, 37°C. After 2 hours temperature was lowered to 25° C. and incubationcontinued for additional one hour. Protein expression was induced byadding IPTG to final concentration 0.1 mM and the culture was incubatedat 25° C. for four hours. Cells were collected by centrifugation(Beckman JLA 8.1000, 20 min., 12.000 g) and washed in PBS buffer (140 mMNaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄ adjusted to pH 7.4).

Lysis and Lysate Clearance.

Bacterial pellet was resuspended in PBS supplemented with 1 mg/mllysozyme, 10 U/ml DNase, 5 mM MgCl₂. After 30 minutes of incubation onice, bacteria were subjected to sonication (12 min total, pulse 1 sec: 2sec=on: off) with constant stirring on ice. Bacterial lysate wasclarified by centrifugation at 50.000×g, 4° C. for 30 minutes (Beckman,Type 45 Ti) and subsequent ultracentrifugation at 180.000×g, 4° C. for 1hour (Beckman, Type 60 Ti).

Chromatography.

Clarified bacterial lysate (80 ml) was applied on HiPrep Heparin 16/10column by a sample pump in the direct load mode. Column was extensivelywashed with 20% B and target protein was eluted by 60% B step (FIG. 11).Due to high target protein content in bacterial lysate, capture onHiPrep Heparin 16/10 column was repeated twice.

In FIG. 11, representing the purification of the mouse TDG on HiPrepHeparin 16/10 column, A stands for elution by PBS and B stands forelution by PBS, 1M NaCl. The flow rate was 5 ml/min.

Eluted protein fractions were pooled and applied on GSTPrep 16/10column. The column was washed by PBS buffer and buffer was changed forCleavage buffer pH 8.8 (20 mM Tris-Cl pH 8.8, 50 mM NaCl, 1 mM EDTA, 1mM DTT). Even though pH value of this buffer is higher than recommendedfor PreScission Protease, tests on parallel GSTrap 5 ml columins showedno difference in cleavage efficiency of GST::TDG when buffers of pH 8.0and pH 8.8 were compared (data not shown). PreScission Protease (400 U)in 7 ml of Cleavage buffer pH 8.8 was injected on the GSTPrep 16/10column by the sample pump in the direct load mode, taking inconsideration dead volume of the pump and sample tubing (about 2.5 ml).After overnight incubation at 10° C., cleaved product was collected in36 ml of the Cleavage buffer pH 8.8 using GSTrap 5 ml auxiliary columndownstream of the GSTPrep 16/10 to eliminate GST and PreScissionProtease leakage. The uncleaved protein was eluted by GSH elution buffer(50 mM Tris-HCl pH 8.0, 10 mM reduced glutathione) (FIG. 12).

In FIG. 12, representing purification of the mouse GST::TDG fusionprotein on the GSTPrep 16/10 column, A stands for elution by PBS,Cleavage buffer pH 8.8; and B stands for elution by glutathione elutionbuffer. Flow rate: 2 ml/min.

Protein from the on-column cleavage was applied on Resource Q 6 mlcolumn equilibrated in 5% B (A: 20 mM Tris-Cl pH 8.8, B: 20 mM Tris-ClpH 8.8, 1M NaCl), washed by 10% B and 10%-25% B gradient in 40 columnvolumes was applied. Fractions from the major peak were collected,diluted with buffer A and re-applied on the same column in order toconcentrate the sample by 40% B step prior to gel filtration (data notshown).

Half of the concentrated TDG from Resource Q 6 ml column (2 ml) wasinjected on HiLoad 16/60 Superdex 200 prep grade column and fractionatedin buffer containing 20 mM Tris-Cl pH 8.0, 150 mM NaCl, 1 mM DTT and 0.1mM EDTA at 1 ml/min.

In FIG. 13, representing the purification of mouse TDG on HiLoad 16/60Superdex 200 prep grade column, the arrows indicate elution volumes ofthe standard proteins from the Gel Filtration LMW and HMW CalibrationKits.

Fractions from the major peak were concentrated to volume 300 μl andconcentration 22 mg/ml. Protein sample was analyzed by MALDI-TOF/MS inorder to confirm identity of the purified protein.

Calculated Mw: 178 kDa (4×44 kDa=176 kDa)

FIG. 14 represents SDS-PAGE analysis of the protein samples from mouseTDG purification K) Bacterial lysate, (L) Flow through HiPrep Heparin16/10 column, (M) 60% B elution from HiPrep Heparin 16/10 column, (N)Flow through GSTPrep 16/10 column, (O) on-column cleaved product, (P)glutathione elution, (O) protein purified by salt gradient on Resource Q6 ml column, (R) concentrated protein by 40% B step on Resource Q 6 mlcolumn, (S) concentrated fractions from HiLoad 16/60 Superdex 200 prepcolurmn.

The modification of the TDG protein can be post-translationally modifiedto confer a specific biological function. U. Hardeland, R. Steinacher,J. Jiricny and P. Schar. EMBO J. 2002 21(6) p.1456-64 (Modification ofthe human thymine-DNA glycosylase by ubiquitin-like proteins facilitatesenzymatic turnover) describe the biologically relevant modification. Theproduction of the specific modified TDG is important for selectedapplications, and the production of suitable amounts of the modifiedtarget is a difficult task. The method provided by the invention allowsfor the post-translational modification of endogenous and/or recombinantTDG and using either programmed lysate, reconstituted enzymaticcomponents, and/or a combination of the two, can provide a sumoylationpost-translational modifying event to the immobilized TDG molecule.

1. A method for post translational modification of a protein orpolypeptide in the presence of a modifying composition capable ofproviding at least one modification comprising brining a liquid phaseincluding the protein or polypeptide and a liquid extract of eukaryoteor prokaryote cells into contact with a solid phase to immobilize theprotein or polypeptide, and bringing the resultant solid phase carryingthe immobilized protein or polypeptide at least once into contact with aliquid phase including the modifying composition to permit suchmodification to occur.
 2. The method of claim 1, wherein the protein orpolypeptide is a recombinant protein or polypeptide produced in a hostcell.
 3. The method of claim 1, wherein the extract is brought intocontact with the solid phase without preliminary purification.
 4. Themethod of claim 1, wherein the modifying composition includes at leastone enzyme capable of catalyzing a modification reaction of the proteinor polypeptide and any other components necessary for modifying theprotein or polypeptide.
 5. The method of claim 1, further comprisingfreeing the modified protein or polypeptide from the solid phase andcollecting the freed protein or polypeptide by bringing the solid phasecarrying the modified immobilized protein or polypeptide into contactwith a liquid phase.
 6. The method of claim 1, wherein the liquid phaseincluding the composition capable of modifying the protein orpolypeptide is brought into contact with the solid phase carrying theimmobilized protein or polypeptide more than once.
 7. The method ofclaim 6, wherein the liquid phase comprising the composition capable ofmodifying the protein or polypeptide is recirculated in contact with thesolid phase carrying the immobilized protein or polypeptide.
 8. Themethod of claim 1, wherein the protein or polypeptide is associated witha moiety capable of selective immobilization on the solid phase.
 9. Themethod of claim 1, wherein the solid phase is an affinity resin.
 10. Themethod of claim 9, wherein the protein or polypeptide is a recombinantprotein fused to a protein or peptide fragment having affinity for thesolid phase.
 11. The method of claim 1, wherein the solid phase is anion exchange resin
 12. The method of claim 1, wherein the modificationof the protein or polypeptide is selected from the group consisting ofacylation, phosphorylation, dephosphorylation, SUMOylation,ubiquitinylation, carboxymethylation, formylation, acetylation,deacetylation, gamma carboxyglutamic acid, norleucine, amidation,deamidation, carboxylation, carboxyamylation, sulfation, methylation,demethylation, hydroxylation, ADP-ribosylation, maturation, adenylation,O-linked glycosylation, N-linked glycosylation, methonine oxidation, andaddition of lipid (prenylation).
 13. The method of claim 12, wherein thecomposition capable of modifying the protein or polypeptide includes atleast one kinase.
 14. The method of claim 12, wherein the protein orpolypeptide includes at least one source of SUMO protein, wherein thecomposition capable of modifying the protein or polypeptide includes atleast one enzyme capable of catalyzing SUMOylation.
 15. The method ofclaim 12, wherein the protein or polypeptide has at least one lysineresidue, in the presence of at least one source of ubiquitin, whereinthe composition capable of modifying the protein or polypeptide includesat least one of the enzymes of the ubiquitination multi-enzyme system.16. The method of claim 12, wherein the protein or polypeptide has atleast one cysteine residue, and the composition capable of modifying theprotein or polypeptide includes a carboxymethylase.
 17. The method ofclaim 12, wherein the protein or polypeptide includes at least oneresidue selected from a lysine residue and an arginine residue, and thecomposition capable of modifying the protein or polypeptide comprises anacetyltransferase or a methyltransferase.
 18. The method of claim 12,wherein the composition capable of modifying the protein or polypeptideincludes an acyltransferase/amidase.
 19. (cancelled)
 20. The method ofclaim 12, wherein the protein or polypeptide includes at least oneresidue selected from the group consisting of an asparagine residue inthe sequence Asn-X-Ser/Thr, a serine residue and a threonine residue andthe composition capable of modifying the protein or polypeptidecomprises a glycosylase.
 21. A kit for modification of a protein orpolypeptide, comprising components for immobilizing the protein orpolypeptide on a solid phase and at least one component for modifyingthe immobilized protein.
 22. The kit of claim 21, including componentsfor freeing and collecting the modified protein or polypeptide from thesolid phase. 23-25. (canceled)